A growing body of evidence suggests that there is a role for inflammatory pathways, initiated by the innate immune system, in cardiovascular disease. However, the mechanisms by which these innate immune pathways participate in heart disease have not been elucidated. Our long-term goal is to elucidate the immunoregulatory and pro-inflammatory signaling mechanisms associated with myocardial ischemia/reperfusion (I/R) injury. This proposal focuses on the role of Toll-like receptor (TLR) mediated signaling pathways in myocardial I/R injury. Toll receptors are an ancient and evolutionarily conserved family of signal transducing molecules which are critical for the induction of innate immunity and inflammation. The central hypotheses are: i) TLR mediated signaling pathways play a pivotal role in regulating immune and inflammatory responses following I/R and ii) modulation of specific TLR signaling pathways will protect the myocardium from I/R injury. The rationale for the proposed studies is that we have preliminary data suggesting that TLR signaling pathways play an important role in myocardial I/R injury. To critically evaluate the hypotheses we will pursue four specific aims. Specific Aim 1. Define the role of TLR mediated MyD88-dependent signaling in myocardial I/R injury. We will determine whether inhibition of TLR mediated MyD88-dependent signaling enhances or suppresses cardiac injury and functional recovery. Specific Aim 2. Investigate the role of TLR mediated PI3K/Akt signaling in myocardial I/R injury. PI3K/Akt signaling is a TLR mediated pathway, that does not signal through MyD88. We will determine whether activation or inhibition of PI3K activity will protect or enhance myocardial I/R injury. Specific Aim 3. Determine the effects of modulation of TLR mediated MyD88-dependent and PI3K/Akt pathways on myocardial injury following I/R. We will determine whether simultaneously blocking TLR mediated MyD88- dependent pathway and activating PI3K/Akt signaling could result in maximal cardioprotection. Specific Aim 4. Elucidate the cardioprotective mechanisms of TLR agonists. LPS, a TLR4 agonist requires at least 8 hrs of pretreatment to induce cardioprotection. Glucan phosphate, a TLR2 agonist, rapidly protects the heart from I/R injury when administered immediately before or after ischemia. We will employ these TLR specific agonists as tools to dissect the immune signaling pathways which are critical to I/R injury.